PROJECT SUMMARY Chronic neuropathic pain is difficult to treat and remains a major clinical problem despite numerous clinical and preclinical studies. Our long-term goal is to identify new targets for treatment/prevention of chronic neuropathic pain. Mounting evidence suggests that epigenetic mechanisms and regulation via microRNAs (miRs) are associated with the transition from acute to chronic pain. The goal of this project is to identify REST- and G9a- related transcriptomic and epigenomic regulatory networks in the dorsal root ganglion (DRG) and their roles in the development of neuropathic pain. The transcriptional repressor REST is a major epigenomic regulator. In nerve injury-induced neuropathic pain, epigenetic silencing by REST overexpression in DRG neurons has been implicated in the repression of targets such as Oprm1, Kcnd3, Kcnq2, and scn10a genes. Histone methyl-transferase G9a is also an epigenomic corepressor of many transcription factors including REST. We recently found that G9a is critical in the epigenetic silencing of almost all K+ channel genes in DRG neurons via increased histone H3K9me2 on the genes' promoters during acute-to-chronic pain transition. Accordingly, mice with G9a ablated from DRG neurons do not develop chronic pain after nerve injury. Thus, both REST and G9a present suitable molecular probes to decipher the genetic and epigenetic bases of chronic pain. However, knowledge is limited about the transcriptomic and epigenomic networks associated with REST and G9a in neuropathic pain development. This multiple-PI grant is a collaboration between two labs with complementary expertise. In addition to the G9a mouse model described above, the two labs have developed an innovative experimental system consisting of complementary Rest conditional knock-out (cKO) and new conditional human REST overexpression (cOE) mouse models. Preliminary results indicate that whereas DRG-specific Rest cKO mice show attenuated pain hypersensitivity after nerve injury, DRG-specific REST cOE mice exhibit pain hypersensitivity. Thus, the two contrasting mouse models recapitulate the chronic pain transition, as anticipated based on work in the field, and provide an in vivo system to study transcriptional mechanisms of chronic pain. Here we propose to the test the central hypothesis that REST and G9a are involved in the development of chronic pain after DRG nerve injury via unique transcriptomic and epigenomic signatures. If successful, our proposed research will identify all REST and G9a targets in DRG neurons including mRNAs and miRs, as well as REST- and G9a-mediated epigenomic changes in the development of chronic pain after nerve injury. We will also functionally identify those REST- and G9a-regulated miRs that could be utilized to block REST- and G9a-mediated chronic pain. We will compare our results to those obtained in chemotherapy- induced chronic pain. In summary, the results from this work are likely to generate new signatures and biomarkers that could potentially be utilized to predict patient susceptibility or resistance to chronic pain development. The results could also be used to identify new actionable drug targets in chronic pain.